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Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.

Behfar A, Perez-Terzic C, Faustino RS, Arrell DK, Hodgson DM, Yamada S, Puceat M, Niederländer N, Alekseev AE, Zingman LV, Terzic A - J. Exp. Med. (2007)

Bottom Line: Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart.Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny.Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

View Article: PubMed Central - PubMed

Affiliation: Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-alpha to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-alpha-induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

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Tumorigenic risk of embryonic stem cell therapy. (A–C) Transplantation of 3 × 105 embryonic stem cells into normal heart (A, transverse section) generated α-actinin–positive cyan fluorescent embryonic stem cell–derived cardiomyocytes (B and inset) that properly integrated into host myocardium (C). Bars: (A) 2 mm; (B) 70 μm; (B, inset and C) 10 μm. (D and E) Embryonic stem cells injected at 106–3 × 106 cells per heart harbored a risk for uncontrolled growth with formation of teratoma that remained encapsulated (D and inset) or protruded into the thoracic cavity (E). Bars: (D and E) 2 mm; (D inset) 300 μm. (F) On histology with hematoxylin-eosin staining, diverse embryonic stem cell–derived phenotypes were documented. These included osteoblasts, chondrocytes, endothelial, epithelial, and germ cell types (first row), and adipocytes, keratinocytes, and myoblasts (third row), reflecting embryonic stem cell pluripotency. Immunohistochemistry was used to verify the multiplicity of cell types, i.e., using SOX9 to confirm chondrocytes and cytokeratin 7 for acinar epithelium (second row). Moreover, tumors derived from embryonic stem cells programmed to express GFP driven by the TIE2 promoter visualized the endothelial phenotype (second row), whereas embryonic stem cells engineered to express CFP under the cardiac actin promoter revealed the presence of embryonic stem cell–derived cardiomyocytes within teratomas (second and third row). Bars: F (all panels except epithelial in first row) 10 μm; (epithelial in first row) 5 μm; (CFP, inset) 100 μm.
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fig1: Tumorigenic risk of embryonic stem cell therapy. (A–C) Transplantation of 3 × 105 embryonic stem cells into normal heart (A, transverse section) generated α-actinin–positive cyan fluorescent embryonic stem cell–derived cardiomyocytes (B and inset) that properly integrated into host myocardium (C). Bars: (A) 2 mm; (B) 70 μm; (B, inset and C) 10 μm. (D and E) Embryonic stem cells injected at 106–3 × 106 cells per heart harbored a risk for uncontrolled growth with formation of teratoma that remained encapsulated (D and inset) or protruded into the thoracic cavity (E). Bars: (D and E) 2 mm; (D inset) 300 μm. (F) On histology with hematoxylin-eosin staining, diverse embryonic stem cell–derived phenotypes were documented. These included osteoblasts, chondrocytes, endothelial, epithelial, and germ cell types (first row), and adipocytes, keratinocytes, and myoblasts (third row), reflecting embryonic stem cell pluripotency. Immunohistochemistry was used to verify the multiplicity of cell types, i.e., using SOX9 to confirm chondrocytes and cytokeratin 7 for acinar epithelium (second row). Moreover, tumors derived from embryonic stem cells programmed to express GFP driven by the TIE2 promoter visualized the endothelial phenotype (second row), whereas embryonic stem cells engineered to express CFP under the cardiac actin promoter revealed the presence of embryonic stem cell–derived cardiomyocytes within teratomas (second and third row). Bars: F (all panels except epithelial in first row) 10 μm; (epithelial in first row) 5 μm; (CFP, inset) 100 μm.

Mentions: To assess the tumorigenic risk of pluripotent stem cells differentiating outside of the natural embryonic program, embryonic stem cells were delivered at increasing loads into the myocardial parenchyma of wild-type mice. Delivery of embryonic stem cells at ≤1,000 cells/mg of myocardial tissue (∼3 × 105 stem cells/heart) resulted in incorporation of stem cell–derived cardiomyocytes in the area of transplantation (n = 50 mice), tracked by fluorescence emitted upon cardiac differentiation (Fig. 1, A and B). Autofluorescence and cell fusion were excluded through multiwavelength immunocytochemical and nuclear probing, indicating that embryonic stem cells undergo nonfusogenic cardiac transformation in the recipient heart (Fig. 1 C). Transplantation of 3,000 embryonic stem cells/mg (∼106 stem cells/heart) resulted in teratoma formation within the myocardial parenchyma in 18% of treated hearts (Fig. 1 D). Delivered at 10,000 per mg of myocardial mass (∼3 × 106 stem cells/heart), embryonic stem cells escaped cardiogenic differentiation in 68% of treated animals, generating massive tumors emanating from the heart into the thoracic cavity (Fig. 1 E). Embryonic stem cell–derived teratoma consisted of a multigerminal cellular heterogeneity, including osteoblasts, chondrocytes, adipocytes, keratinocytes, myoblasts, endothelial, epithelial tissue, and germinal cells (Fig. 1 F). Verification of cytotypes was made by immunostaining with SOX9 (chondrocytes) and cytokeratin7 (acinar epithelial cells) and through delivery of cells engineered to express cyan fluorescent protein (CFP) under control of the cardiac actin promoter (for cardiac cells) or GFP under control of the Tie2 promoter (for endothelial cells), revealing the narrow margin of safety associated with delivery of pluripotent stem cells in wild-type hearts (Fig. 1 F).


Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.

Behfar A, Perez-Terzic C, Faustino RS, Arrell DK, Hodgson DM, Yamada S, Puceat M, Niederländer N, Alekseev AE, Zingman LV, Terzic A - J. Exp. Med. (2007)

Tumorigenic risk of embryonic stem cell therapy. (A–C) Transplantation of 3 × 105 embryonic stem cells into normal heart (A, transverse section) generated α-actinin–positive cyan fluorescent embryonic stem cell–derived cardiomyocytes (B and inset) that properly integrated into host myocardium (C). Bars: (A) 2 mm; (B) 70 μm; (B, inset and C) 10 μm. (D and E) Embryonic stem cells injected at 106–3 × 106 cells per heart harbored a risk for uncontrolled growth with formation of teratoma that remained encapsulated (D and inset) or protruded into the thoracic cavity (E). Bars: (D and E) 2 mm; (D inset) 300 μm. (F) On histology with hematoxylin-eosin staining, diverse embryonic stem cell–derived phenotypes were documented. These included osteoblasts, chondrocytes, endothelial, epithelial, and germ cell types (first row), and adipocytes, keratinocytes, and myoblasts (third row), reflecting embryonic stem cell pluripotency. Immunohistochemistry was used to verify the multiplicity of cell types, i.e., using SOX9 to confirm chondrocytes and cytokeratin 7 for acinar epithelium (second row). Moreover, tumors derived from embryonic stem cells programmed to express GFP driven by the TIE2 promoter visualized the endothelial phenotype (second row), whereas embryonic stem cells engineered to express CFP under the cardiac actin promoter revealed the presence of embryonic stem cell–derived cardiomyocytes within teratomas (second and third row). Bars: F (all panels except epithelial in first row) 10 μm; (epithelial in first row) 5 μm; (CFP, inset) 100 μm.
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fig1: Tumorigenic risk of embryonic stem cell therapy. (A–C) Transplantation of 3 × 105 embryonic stem cells into normal heart (A, transverse section) generated α-actinin–positive cyan fluorescent embryonic stem cell–derived cardiomyocytes (B and inset) that properly integrated into host myocardium (C). Bars: (A) 2 mm; (B) 70 μm; (B, inset and C) 10 μm. (D and E) Embryonic stem cells injected at 106–3 × 106 cells per heart harbored a risk for uncontrolled growth with formation of teratoma that remained encapsulated (D and inset) or protruded into the thoracic cavity (E). Bars: (D and E) 2 mm; (D inset) 300 μm. (F) On histology with hematoxylin-eosin staining, diverse embryonic stem cell–derived phenotypes were documented. These included osteoblasts, chondrocytes, endothelial, epithelial, and germ cell types (first row), and adipocytes, keratinocytes, and myoblasts (third row), reflecting embryonic stem cell pluripotency. Immunohistochemistry was used to verify the multiplicity of cell types, i.e., using SOX9 to confirm chondrocytes and cytokeratin 7 for acinar epithelium (second row). Moreover, tumors derived from embryonic stem cells programmed to express GFP driven by the TIE2 promoter visualized the endothelial phenotype (second row), whereas embryonic stem cells engineered to express CFP under the cardiac actin promoter revealed the presence of embryonic stem cell–derived cardiomyocytes within teratomas (second and third row). Bars: F (all panels except epithelial in first row) 10 μm; (epithelial in first row) 5 μm; (CFP, inset) 100 μm.
Mentions: To assess the tumorigenic risk of pluripotent stem cells differentiating outside of the natural embryonic program, embryonic stem cells were delivered at increasing loads into the myocardial parenchyma of wild-type mice. Delivery of embryonic stem cells at ≤1,000 cells/mg of myocardial tissue (∼3 × 105 stem cells/heart) resulted in incorporation of stem cell–derived cardiomyocytes in the area of transplantation (n = 50 mice), tracked by fluorescence emitted upon cardiac differentiation (Fig. 1, A and B). Autofluorescence and cell fusion were excluded through multiwavelength immunocytochemical and nuclear probing, indicating that embryonic stem cells undergo nonfusogenic cardiac transformation in the recipient heart (Fig. 1 C). Transplantation of 3,000 embryonic stem cells/mg (∼106 stem cells/heart) resulted in teratoma formation within the myocardial parenchyma in 18% of treated hearts (Fig. 1 D). Delivered at 10,000 per mg of myocardial mass (∼3 × 106 stem cells/heart), embryonic stem cells escaped cardiogenic differentiation in 68% of treated animals, generating massive tumors emanating from the heart into the thoracic cavity (Fig. 1 E). Embryonic stem cell–derived teratoma consisted of a multigerminal cellular heterogeneity, including osteoblasts, chondrocytes, adipocytes, keratinocytes, myoblasts, endothelial, epithelial tissue, and germinal cells (Fig. 1 F). Verification of cytotypes was made by immunostaining with SOX9 (chondrocytes) and cytokeratin7 (acinar epithelial cells) and through delivery of cells engineered to express cyan fluorescent protein (CFP) under control of the cardiac actin promoter (for cardiac cells) or GFP under control of the Tie2 promoter (for endothelial cells), revealing the narrow margin of safety associated with delivery of pluripotent stem cells in wild-type hearts (Fig. 1 F).

Bottom Line: Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart.Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny.Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

View Article: PubMed Central - PubMed

Affiliation: Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-alpha, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-alpha to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-alpha-induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

Show MeSH
Related in: MedlinePlus